The main objective of vaccination is to induce a specific and effective immune response against a pathogen that also produces protection against infection and/or a disease and results in its elimination. The concept that the immune response against a particular antigen can be improved by the addition of certain compounds in the vaccine formulation was shown about 100 years ago, when the aluminum salts were introduced into the formulations and were called “adjuvant” (G. Leroux-Roels (2010) Vaccine 28S C25-C36).
Traditional vaccines consist of inactivated or attenuated pathogens, or toxins derived from these microorganisms. Although the use of inactivated or attenuated pathogens has high immunogenicity, currently is unattractive in vaccinology because of the high toxicity of these preparations.
Therefore, there is growing interest in adjuvants in the research related to the development of new generations of vaccines based on recombinant protein subunits, synthetic peptides and deoxyribonucleic acid (DNA) plasmids. These new vaccine variants, although less toxic, are mostly less immunogenic when they are administered without an immunostimulatory adjuvant. Due to this reason, in recent years has increased the need for safer and more potent adjuvants (Saenz et al. (2010) Vaccine 28 (47): 7556-7562).
Although the use of adjuvant is well known, the manner in which they act is less clear. It is considered that, in general, they increase the effectiveness of vaccines through several mechanisms including: 1) increased antigen processing and presentation by dendritic cells, 2) induction of a “danger” signal by signaling pathways mediated by receptors that recognize patterns associated pathogens, such as the “Toll” receptors, and 3) by activating co-stimulatory signals that activate lymphocytes. These mechanisms are triggered by cytokine induction and up-regulation of the expression of appropriate co-stimulatory signals (Secombes (2010) Fish & Shellfish Immunology, 409-416).
Several endogenous molecules and proteins (Yin and Kwang (2000) Fish & Shellfish Immunology 10, 375-378; Lingnau et al. (2007) Expert Rev Vaccines 6 (5): 741-6; Zhang et al. (2010) Vaccine 28: 5114-5127) have been studied as adjuvants. Also, it is known that certain immunostimulating peptides can act as an adjuvant in vivo, stimulating the immune response of protein or peptide antigens (Saenz et al. (2010) Vaccine 28 (47): 7556-7562).
PACAP belongs to the superfamily of secretin/glucagon/vasoactive intestinal peptide (Miyata et al (1989) Biochem Biophys Res Commun 164: 567-574). It is a multifunctional neuropeptide which plays important roles as neurotrophic and hypophysiotropic factor, as a neurotransmitter, neuromodulator and vasodilator molecule in mammals (Arimura A. (1998) Japanese Journal of Physiology 48: 301-31). It has been demonstrated its role in the cell division and differentiation and also in the cell death (Sherwood et al. (2000) Endocrine Review 21: 619-670). This peptide exists in two molecular forms of 38 (PACAP38) and 27 (PACAP27) amino acids (Miyata et al. (1990) Biochemical and Biophysical Research Communications 170:643-8). The biological actions of PACAP are exerted through a family of three VIP/PACAP receptors that belong to the secretin G-protein-coupled receptor: the type I receptor, which is highly specific for PACAP and is named as PAC-1; and type II receptors, which exhibit the same affinity for PACAP than for vasoactive intestinal peptide (VIP), which are known as VPAC-1 and VPAC-2 (Vaudry et al. (2000) Pharmacol Rev 52: 269-324).
PACAP is widely distributed in various tissues, including those related to the immune system, although the presence of this peptide and its receptors in cells of the immune system in mammals has only been partially elucidated (Gaytan et al. (1994) Cell Tissue Res 276:223-7, Abad et al. (2002) Neuroimmunomodulation 10:177-86). PACAP modulates the inflammatory response through the regulation of the interleukin-6 (IL-6) and interleukin-10 (IL-10) (Martinez et al. (1996) J Immunol 156 (11):4128-36; Martinez et al. (1998) J Neuroimmunol 85 (2): 155-67), Martinez et al. (1998) J Leukoc Biol 63 (5): 591-601).
In activated macrophages, PACAP inhibits the production of pro-inflammatory cytokines and stimulates the production of anti-inflammatory cytokines, thus allowing the homeostasis of the immune system. Additionally, it is known that PACAP reduces the expression of the co-stimulatory molecules B7.2/B7.1 and the subsequent activation of T helper cells (Th). On the other hand, PACAP inhibit in activated macrophages the production of IL-6 through its PAC-1 receptor, suppressing inflammation (Martinez et al. (1998) J Neuroimmunol 85 (2): 155-67, Martinez et al. (1998) J Leukoc Biol 1998, 63 (5): 591-601). The inhibitory action of PACAP over IL-6 transcription in response to intense inflammatory stimuli helps the tissue protection and the immune system homeostasis (Martinez et al. (1998) J Neuroimmunol 85(2):155-67; Martinez et al. (1998) J Leukoc Biol. 1998 May; 63(5):591-601). In contrast, PACAP induce the expression of B7.2 and promotes cellular differentiation to Th2 in non-stimulated macrophages (Delgado and Ganea (2001) Arch Immunol Ther Exp (Warsz) 49(2):101-10). The presence of PACAP in the lymphoid organs of ducks has been reported (Squillacioti et al. (2005) Anatomia, Histologia, Embryologia. Volume 34, Issue Supplements 1, page 49).
The use of PACAP in mammals, as a therapeutic agent for treating autoimmune diseases such as septic shock, rheumatoid arthritis and Crohn's disease, has been proposed (Gomariz et al. (2006) Ann. NY Acad. Sci 1070: 51-74). It is known that in autoimmune diseases there is an uncontrolled immune response against substances and tissues of the organism. In this regard, PACAP prevents inflammation in animal models of autoimmune diseases, through a proper balance of cytokines and chemokines and its receptors, through the recruitment of immune cells and by regulating the generation and activation of Th1 cells and the cytokines which these cells secrete.
Moreover, it is known that molecules involved in the immune system homeostasis, such as “Toll” receptors, are essential for activation of the innate immune response, which boosts the adaptive immune response. However, this response may lead to the pathogenesis of acute and/or chronic inflammation, autoimmunity, and cancer (Gomariz et al. (2010) Current Pharmaceutical Design 16: 1063-1080). In this regard, it is known that the VIP-PACAP system is involved in the regulation of the expression of genes encoding these receptors (Gomariz et al. (2006) Ann. NY Acad. Sci 1070: 51-74). Therefore, both peptides may induce disturbances in the regulation pathways of production of these receptors.
PACAP attenuates the circulating levels of cytokines mediated by the endogenous molecule HMGB1 (high mobility group box 1) (Tang et al. (2008) International Immunopharmacology 8 (12): 1646-1651), and inhibits their release. It is known that HMGB1 molecule and its derived peptides are capable of acting as adjuvant, enhancing the immune response against a peptide antigen and protein (Saenz et al. (2010) Vaccine 28 (47): 7556-7562).
The knowledge about the function of PACAP in the modulation of the fish immune response is limited to studies performed by our research group. We demonstrated that the recombinant Clarias gariepinus PACAP administrated by immersion baths or by injection not only promotes growth but also stimulates innate immune parameters (lysozyme, nitric oxide derived metabolites and antioxidant defenses) and acquired immunity (IgM) in larvae and juveniles fish (Carpio et al. (2008) Fish and Shellfish Immunology 25:439-45; Lugo et al. (2010) Fish and Shellfish Immunology 29:513-520). These properties of PACAP were described in the international patent application WO2007/059714, “Neuropéptidos para el cultivo de organismos acuáticos”.
At present, in the field of human and veterinary medicine, there is still interest in identifying compounds that may be used as safe and more potent adjuvants that may be incorporated into existing vaccines, and in those which are under development.